Analiza N-glikana lizosomalnih membranskih glikoproteina modela Niemann-Pickove bolesti tip C1

master's thesis

• Title (english): Analysis of N-glycans from lysosomal membrane glycoproteins of Niemann-Pick type C1 model
• Author: Tena Cupar
• Mentor: Gordan Lauc (mentor)
• Committee member: Gordan Lauc (predsjednik povjerenstva)
• Committee member: Miranda Sertić (član povjerenstva)
• Committee member: Olga Gornik (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2016-09-12, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Niemann-Pickova bolest tip C1 je rijetka, nasljedna i zasad neizlječiva lizosomska bolest nakupljanja. Glavna odlika bolesti je nakupljanje kolesterola u endolizosomalnom susutavu. Uzrok bolesti je mutacija proteina NPC1 koji je potreban za izlazak kolesterola iz lizosoma. Bolest je teška i progresivna i uključuje neurološke simptome kao što su ataksija, disfagija i demencija. U većini slučajeva javlja se i (hepato)splenomegalija. Osim toga, Niemann-Pickova bolest tip C je slična Alzheimerovoj budući da se u obje bolesti javljaju neurofibrilarni snopići i amiloidogeno procesiranje APP.
Istraživanjem N-glikana membranskih lizosomalnih glikoproteinima pokušalo se pogledati na ovu bolest iz nove perspektive. U tu svrhu korišene su CHO-NPC1 -/- i CHOwt stanice kao kontrola, te principi magnetske kromatografije i fazne separacije detergentom Triton X-114 za izolaciju glikoproteina. N-glikani odvojeni sa glikoproteina i obilježeni sa 2-aminobenzamidom bili su analizirani HILIC-UPLC metodom. Za asignaciju pojedinih kromatografskih vršaka korištena je masena spektrometrija i usporedba s bazom podataka GlycoBase.
Otkrivene su statistički značajne razlike u N-glikomu lizosomalne membrane između CHO NPC-/- i CHOwt. Primijećen je porast kompleksnih struktura N-glikana, kao i povećana količina M5 glikana na lizosomalnim membranskim proteinima NPC modela. Također je primijećena razlika u broju manoza na oligomanoznim glikanima. Ova otkrića potvrdila su neka od otprije provedenih istraživanja na temu NPC.

Abstract (english)

Niemann-Pick's disease type C1 is a rare, inheritable and currently untreatable lysosomal storage disease. The main characteristic of this disease is accumulation of cholesterol in the endo-lysosomal system. The cause of the disease is a mutation in the NPC1 protein, which is necessary for egress of cholesterol from lysosomes. The disease is severe and progressive and includes neurological symptoms such as ataxia, dysphagia and dementia. In most cases, (hepato)splenomegaly is also present. Besides, Niemann-Pick's disease type C is simmilar to Alzheimer's disease. Neurofibrillary tangles and amyloidogenic processing of APP are present in both of these conditions.
An effort was made to see this disease in a new light by investigating the N-glycans of lysosomal membrane glycoproteins. CHO-NPC1 -/- cell culture was used for this purpose, as well as CHOwt cells as a control group. In order to isolate the lysosomal membrane glycoproteins, magnetic chromatography and Triton x-114 mediated phase separation were used. The N-glycans were separated from the proteins and labeled with 2-aminobenzamide and then analysed with HILIC-UPLC. Peak assignment was made with mass spectrometry and GlycoBase database.
Statistically significant differences were discovered in the N-glycome of the lysosomal membrane. There was a significant increase in complex glycan structures, as well as an increase of M5 type glycans. There was also a difference mannose residues number of oligomannose glycans. M8 glycan was more abundant in CHOwt cells. Those discoveries were in line with some of the previous work done on the subject.

• Keywords: N-glikan; lizosom; lizosomalna membrana; UPLC-HILIC; Niemann-Pickova bolest tip C1; NPC1
• Keywords (english): N-glycans; lysosome; lysosomal membrane; UPLC-HILIC; Niemann-picks' disease type C1; NPC1
• Language: croatian
• URN:NBN: urn:nbn:hr:163:318128
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2016-11-30 16:39:20